Chair, Department of Chemical and Systems Biology, Stanford University School of Medicine (2016 - Present)
Executive Committee Member, ChEM-H Institute (2012 - Present)
Director, Advisory Committee for the Scholarly Concentrations Program in the Molecular Basis of Medicine (2012 - Present)
Faculty Director, Stanford High-Throughput Bioscience Center (2003 - Present)
Member, Medical Scientist Training Program Admissions Committee (2004 - Present)
Executive Committee Member, Stanford University School of Medicine Faculty Senate (2012 - 2016)
Alternate Member, Stanford University Administrative Panel on Laboratory Animal Care (2012 - 2014)
Departmental Representative, Stanford Biosciences Committee on Graduate Admissions and Policy (2012 - 2014)
Honors & Awards
NSF INSPIRE Award, NSF (2013-2017)
Innovation Award, Alex's Lemonade Stand Foundation (2013-2015)
Nature SciCafe Award for Outstanding Research Achievement, Nature Biotechnology and Nature Medicine (2009)
NIH Director's Pioneer Award, NIH (2008-2013)
American Cancer Society Research Scholar Award, American Cancer Society (2008-2011)
Brain Tumor Society Award, Brain Tumor Society/Rachel Molly Markoff Foundation (2006-2008)
Astellas USA Foundation Award, Astella USA Foundation (2005-2006)
Terman Fellow, Stanford University (2005-2008)
Basil O'Connor Starter Scholar Research Award, March of Dimes (2005-2007)
Kimmel Scholar Award, Sidney Kimmel Foundation for Cancer Research (2004-2006)
W. Barry Wood, Jr. Research Award, Johns Hopkins School of Medicine (2003)
American Cancer Society Postdoctoral Fellowship, American Cancer Society (2002-2003)
Damon Runyon-Walter Winchell Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (1999-2002)
A.B., Harvard College, Chemistry (1991)
Ph.D., Harvard University, Chemistry and Chemical Biology (1999)
Postdoctoral Fellow, Johns Hopkins School of Medicine, Molecular Biology and Genetics (2003)
Current Research and Scholarly Interests
The Chen laboratory integrates synthetic chemistry and developmental biology to investigate the molecular mechanisms that control tissue formation, regeneration, and oncogenic transformation. Our research group is currently focused on three major areas: (1) small-molecule and genetic regulators of Hedgehog signaling; (2) chemical tools for studying tissue patterning at the molecular and systems levels; and (3) zebrafish models of tissue biology.
Our interest in the Hedgehog pathway arises from its critical role in the patterning of multiple tissues such as the neural tube, craniofacial structures, limbs, and somites. Aberrant Hedgehog pathway activation in children and adults is also linked to several cancers, including those of the skin, brain, and blood. Since the cellular events that transduce the Hedgehog signal from the cell surface to the nucleus are not well understood, we are pursuing genetic and small-molecule screens for new Hedgehog pathway modulators with novel modes of action. These studies will provide insights into the basic mechanisms of Hedgehog signal transduction, as well as targets and chemical leads for new therapies.
Our laboratory is also investigating the molecular pathways regulate vertebrate tissue formation, homeostasis, and regeneration. We use primarily the zebrafish as a model organism for these studies, exploiting its rapid ex utero development, amenability to both chemical and genetic perturbations, and optical transparency during embryonic and larval stages. As part of these efforts, we have developed caged antisense oligonucleotides and photoactivatable signaling proteins that afford precise spatiotemporal control of gene function. We have also devised new methods for time-resolved lanthande microscopy, enabling ultrasensitive, autofluorescence-free imaging. In conjunction with genetic approaches, these chemical technologies can open new windows into the molecular mechanisms that control vertebrate development and physiology.
- Chemical and Systems Biology Pizza Talks
CSB 224 (Win, Spr, Sum)
Independent Studies (15)
- Advanced Undergraduate Research
CHEM 190 (Aut, Win, Spr, Sum)
- Directed Instruction/Reading
CHEM 110 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Win, Spr)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Win, Spr)
- Research and Special Advanced Work
CHEM 200 (Aut, Win, Spr, Sum)
- Research in Chemistry
CHEM 301 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Win, Spr)
- Advanced Undergraduate Research
- Prior Year Courses
Basal constriction during midbrain-hindbrain boundary morphogenesis is mediated by Wnt5b and focal adhesion kinase.
Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3beta overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
View details for DOI 10.1242/bio.034520
View details for PubMedID 30305282
Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator.
2018; 9 (1): 4045
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.
View details for DOI 10.1038/s41467-018-06447-z
View details for PubMedID 30279493
Ultrasensitive optical imaging with lanthanide lumiphores
Nat. Chem. Biol.
2018; 14: 15-21
In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.
View details for DOI 10.1038/nchembio.2513
View details for PubMedCentralID PMC5726931
- A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies Nat. Genet. 2018; 50: 460-471
Basal constriction during midbrain-hindbrain boundary morphogenesis is mediated by Wnt5b and Focal Adhesion Kinase
View details for DOI 10.1101/251132
Illuminating developmental biology through photochemistry
NATURE CHEMICAL BIOLOGY
2017; 13 (6): 587-598
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
View details for DOI 10.1038/nchembio.2369
View details for Web of Science ID 000401419300009
View details for PubMedID 28514427
Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action
2017; 6: e25174
View details for DOI 10.7554/eLife.25174
- Discovery of novel determinants of endothelial lineage using chimeric heterokaryons Elife 2017; 6: e23588
Reviving Vibration Energy Harvesting and Self-Powered Sensing by a Triboelectric Nanogenerator
2017; 1 (3): 480–521
View details for DOI 10.1016/j.joule.2017.09.004
- A novel missense variant in the GLI3 zinc finger domain in a family with digital anomalies Am. J. Med. Genet. A 2017; 173: 3221-3225
- Hyaluronic acid synthesis is required for zebrafish tail fin regeneration PLoS One 2017; 12: e0171898
An inducible long noncoding RNA amplifies DNA damage signaling.
2016; 48 (11): 1370-1376
Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.
View details for DOI 10.1038/ng.3673
View details for PubMedID 27668660
View details for PubMedCentralID PMC5083181
Tbx16 regulates hox gene activation in mesodermal progenitor cells.
Nature chemical biology
2016; 12 (9): 694-701
The transcription factor T-box 16 (Tbx16, or Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. However, the mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic. We describe here the use of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identified 124 Tbx16-regulated genes that were expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis and somitogenesis. Unexpectedly, we observed that a loss of Tbx16 function precociously activated posterior hox genes in MPCs, and overexpression of a single posterior hox gene was sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs.
View details for DOI 10.1038/nchembio.2124
View details for PubMedID 27376691
View details for PubMedCentralID PMC4990471
- Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity ACS CHEMICAL BIOLOGY 2016; 11 (1): 53-60
- Control of inflammation by stromal Hedgehog pathway activation restrains colitis Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E7545-E7553
I only have eye for ewe: the discovery of cyclopamine and development of Hedgehog pathway-targeting drugs
NATURAL PRODUCT REPORTS
2016; 33 (5): 595-601
Covering: 1950s to 2015During the 1950s, sheep ranchers in the western United States experienced episodic outbreaks of cyclopic lambs. In this highlight I describe how these mysterious incidents were traced to the grazing of Veratrum californicum wildflowers by pregnant ewes, leading to the discovery of cyclopamine () as a plant-derived teratogen. The precise mechanism of cyclopamine action remained enigmatic for 30 years, until this steroid alkaloid was found to be the first specific inhibitor of Hedgehog (Hh) signalling and a direct antagonist of the transmembrane receptor Smoothened (SMO). In addition to being a valuable probe of Hh pathway function, cyclopamine has been used to demonstrate the therapeutic potential of Hh pathway inhibitors. I discuss the development of SMO antagonists as anticancer therapies and emerging challenges.
View details for DOI 10.1039/c5np00153f
View details for Web of Science ID 000375567400002
View details for PubMedID 26787175
View details for PubMedCentralID PMC4856577
Optochemical Dissection of T-box Gene-Dependent Medial Floor Plate Development.
ACS chemical biology
2015; 10 (6): 1466-1475
In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.
View details for DOI 10.1021/cb5010178
View details for PubMedID 25781211
View details for PubMedCentralID PMC4672996
Thinking big with small molecules
JOURNAL OF CELL BIOLOGY
2015; 209 (1): 7-9
Synthetic chemistry has enabled scientists to explore the frontiers of cell biology, limited only by the laws of chemical bonding and reactivity. As we investigate biological questions of increasing complexity, new chemical technologies can provide systems-level views of cellular function. Here we discuss some of the molecular probes that illustrate this shift from a "one compound, one gene" paradigm to a more integrated approach to cell biology.
View details for DOI 10.1083/jcb.201501084
View details for Web of Science ID 000352960200003
View details for PubMedID 25869661
View details for PubMedCentralID PMC4395478
Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides.
Angewandte Chemie (International ed. in English)
2014; 53 (38): 10114-10118
Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.
View details for DOI 10.1002/anie.201405355
View details for PubMedID 25130695
View details for PubMedCentralID PMC4206551
- Nitroreductase-Activatable Morpholino Oligonucleotides for in Vivo Gene Silencing ACS CHEMICAL BIOLOGY 2014; 9 (9): 1985-1990
Stromal response to Hedgehog signaling restrains pancreatic cancer progression.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (30): E3091-100
Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.
View details for DOI 10.1073/pnas.1411679111
View details for PubMedID 25024225
View details for PubMedCentralID PMC4121834
Arhgap36-dependent activation of Gli transcription factors.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (30): 11061-11066
Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are up-regulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.
View details for DOI 10.1073/pnas.1322362111
View details for PubMedID 25024229
View details for PubMedCentralID PMC4121843
Direct kinetochore-spindle pole connections are not required for chromosome segregation
JOURNAL OF CELL BIOLOGY
2014; 206 (2): 231-243
Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.
View details for DOI 10.1083/jcb.201401090
View details for Web of Science ID 000339462000009
View details for PubMedID 25023516
View details for PubMedCentralID PMC4107786
General method for regulating protein stability with light.
ACS chemical biology
2014; 9 (1): 111-115
Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.
View details for DOI 10.1021/cb400755b
View details for PubMedID 24180414
View details for PubMedCentralID PMC3906921
Nitroreductase-activatable morpholino oligonucleotides for in vivo gene silencing.
ACS chemical biology
2014; 9 (9): 1985–90
Phosphorodiamidate morpholino oligonucleotides are widely used to interrogate gene function in whole organisms, and light-activatable derivatives can reveal spatial and temporal differences in gene activity. We describe here a new class of caged morpholino oligonucleotides that can be activated by the bacterial nitroreductase NfsB. We characterize the activation kinetics of these reagents in vitro and demonstrate their efficacy in zebrafish embryos that express NfsB either ubiquitously or in defined cell populations. In combination with transgenic organisms, such enzyme-actuated antisense tools will enable gene silencing in specific cell types, including tissues that are not amenable to optical targeting.
View details for DOI 10.1021/cb500429u
View details for PubMedID 25069083
- In vivo imaging of Hedgehog pathway activation with a nuclear fluorescent reporter PLoS One 2014; 9: e103661
Post-transcriptional mechanisms contribute to Etv2 repression during vascular development
2013; 384 (1): 128-140
etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.
View details for DOI 10.1016/j.ydbio.2013.08.028
View details for Web of Science ID 000326901100011
View details for PubMedID 24036310
View details for PubMedCentralID PMC4139968
- The stanford institute for chemical biology. ACS chemical biology 2013; 8 (9): 1860-1861
Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage
JOURNAL OF CELL BIOLOGY
2013; 202 (5): 779-792
T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In "frustrated" conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.
View details for DOI 10.1083/jcb.201301004
View details for Web of Science ID 000323870500007
View details for PubMedID 23979719
View details for PubMedCentralID PMC3760611
Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (29): 11976-11981
Centrosome reorientation to the immunological synapse maintains the specificity of T-cell effector function by facilitating the directional release of cytokines and cytolytic factors toward the antigen-presenting cell. This polarization response is driven by the localized accumulation of diacylglycerol, which recruits multiple protein kinase (PK)C isozymes to the synaptic membrane. Here, we used T-cell receptor (TCR) photoactivation and imaging methodology to demonstrate that PKCs control centrosome dynamics through the reciprocal localization of two motor complexes, dynein and nonmuscle myosin (NM)II. Dynein accumulated in the region of TCR stimulation, whereas NMII clustered in the back of the cell, behind the polarizing centrosome. PKC activity, which shaped both dynein and NMII accumulation within this framework, controlled NMII localization directly by phosphorylating inhibitory sites within the myosin regulatory light chain, thereby suppressing NMII clustering in the region of TCR stimulation. Concurrently, phosphorylation of distinct sites within myosin regulatory light chain by Rho kinase drove NMII clustering in areas behind the centrosome. These results reveal a role for NMII in T-cell polarity and demonstrate how it is regulated by upstream signals.
View details for DOI 10.1073/pnas.1306180110
View details for Web of Science ID 000322086100071
View details for PubMedID 23818610
View details for PubMedCentralID PMC3718145
Functional inhibition of UQCRB suppresses angiogenesis in zebrafish.
Biochemical and biophysical research communications
2013; 433 (4): 396-400
As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levels in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.
View details for DOI 10.1016/j.bbrc.2013.02.082
View details for PubMedID 23454382
View details for PubMedCentralID PMC3691074
- Emerging technologies in molecular imaging: new windows into biology. Current opinion in chemical biology 2013
Small-molecule inhibitors of the AAA plus ATPase motor cytoplasmic dynein
2012; 484 (7392): 125-129
The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.
View details for DOI 10.1038/nature10936
View details for Web of Science ID 000302343400047
View details for PubMedID 22425997
View details for PubMedCentralID PMC3321072
The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome
2012; 484 (7392): 115-?
The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.
View details for DOI 10.1038/nature10956
View details for Web of Science ID 000302343400045
View details for PubMedID 22398447
View details for PubMedCentralID PMC3321094
Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development
NATURE CHEMICAL BIOLOGY
2012; 8 (3): 270-276
Transcription factors have diverse roles during embryonic development, combinatorially controlling cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholino oligonucleotides with photoactivatable fluorophores, fluorescence-activated cell sorting and microarray technologies. As a proof of principle, we have dynamically profiled No tail a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies reveal new regulators of notochord development and the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor and demonstrate how optically controlled chemical tools can dissect developmental processes with spatiotemporal precision.
View details for DOI 10.1038/NCHEMBIO.772
View details for Web of Science ID 000300600000012
View details for PubMedID 22286130
View details for PubMedCentralID PMC3288381
Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2012; 51 (28): 6908-6911
Feeling a bit cagey: morpholino-based antisense reagents have been caged through oligonucleotide cyclization, enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined.
View details for DOI 10.1002/anie.201201690
View details for Web of Science ID 000305990800017
View details for PubMedID 22689470
View details for PubMedCentralID PMC3434341
Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons
2011; 138 (23): 5121-5134
In mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.
View details for DOI 10.1242/dev.066159
View details for Web of Science ID 000296769200008
View details for PubMedID 22069186
View details for PubMedCentralID PMC3210494
Hedgehog and retinoic acid signaling cooperate to promote motoneurogenesis in zebrafish
2011; 138 (23): 5113-5119
The precise requirements of Hedgehog (Hh) pathway activity in vertebrate central nervous system development remain unclear, particularly in organisms with both maternally and zygotically derived signaling. Here we describe the motoneural phenotype of zebrafish that lack maternal and zygotic contributions of the Hh signaling transducer Smoothened (MZsmo mutants) and therefore are completely devoid of ligand-dependent pathway activation. Some functional primary motoneurons (PMNs) persist in the absence of Hh signaling, and we find that their induction requires both basal Gli transcription factor activity and retinoic acid (RA) signaling. We also provide evidence that RA pathway activation can modulate Gli function in a Hh ligand-independent manner. These findings support a model in which Hh and RA signaling cooperate to promote PMN cell fates in zebrafish.
View details for DOI 10.1242/dev.066225
View details for Web of Science ID 000296769200007
View details for PubMedID 22069185
Neuropilins are positive regulators of Hedgehog signal transduction
GENES & DEVELOPMENT
2011; 25 (22): 2333-2346
The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.
View details for DOI 10.1101/gad.173054.111
View details for Web of Science ID 000297154700003
View details for PubMedID 22051878
View details for PubMedCentralID PMC3222900
A crucial requirement for Hedgehog signaling in small cell lung cancer
2011; 17 (11): 1504-U1506
Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.
View details for DOI 10.1038/nm.2473
View details for Web of Science ID 000296779300043
View details for PubMedID 21983857
View details for PubMedCentralID PMC3380617
A Small-Molecule Smoothened Agonist Prevents Glucocorticoid-Induced Neonatal Cerebellar Injury
SCIENCE TRANSLATIONAL MEDICINE
2011; 3 (105)
Glucocorticoids are used for treating preterm neonatal infants suffering from life-threatening lung, airway, and cardiovascular conditions. However, several studies have raised concerns about detrimental effects of postnatal glucocorticoid administration on the developing brain leading to cognitive impairment, cerebral palsy, and hypoplasia of the cerebellum, a brain region critical for coordination of movement and higher-order neurological functions. Previously, we showed that glucocorticoids inhibit Sonic hedgehog-Smoothened (Shh-Smo) signaling, the major mitogenic pathway for cerebellar granule neuron precursors. Conversely, activation of Shh-Smo in transgenic mice protects against glucocorticoid-induced neurotoxic effects through induction of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) pathway. Here, we show that systemic administration of a small-molecule agonist of the Shh-Smo pathway (SAG) prevented the neurotoxic effects of glucocorticoids. SAG did not interfere with the beneficial effects of glucocorticoids on lung maturation, and despite the known associations of the Shh pathway with neoplasia, we found that transient (1-week-long) SAG treatment of neonatal animals was well tolerated and did not promote tumor formation. These findings suggest that a small-molecule agonist of Smo has potential as a neuroprotective agent in neonates at risk for glucocorticoid-induced neonatal cerebellar injury.
View details for DOI 10.1126/scitranslmed.3002731
View details for Web of Science ID 000296565100006
View details for PubMedID 22013124
View details for PubMedCentralID PMC3694585
- Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2011
Chemical 'Jekyll and Hyde's: small-molecule inhibitors of developmental signaling pathways
CHEMICAL SOCIETY REVIEWS
2011; 40 (8): 4318-4331
Small molecules that perturb developmental signaling pathways can have devastating effects on embryonic patterning, as evidenced by the chemically induced onset of cyclopic lambs and children with severely shortened limbs during the 1950s. Recent studies, however, have revealed critical roles for these pathways in human disorders and diseases, spurring the re-examination of these compounds as new targeted therapies. In this tutorial review, we describe four case studies of teratogenic compounds, including inhibitors of the Hedgehog (Hh), Wnt, and bone morphogenetic protein (BMP) pathways. We discuss how these teratogens were discovered, their mechanisms of action, their utility as molecular probes, and their potential as therapeutic agents. We also consider current challenges in the field and possible directions for future research.
View details for DOI 10.1039/c1cs15019g
View details for Web of Science ID 000292984900007
View details for PubMedID 21505654
View details for PubMedCentralID PMC3137710
Spatiotemporal Control of Embryonic Gene Expression Using Caged Morpholinos
ZEBRAFISH: GENETICS, GENOMICS AND INFORMATICS, 3RD EDITION
2011; 104: 151-172
Embryonic development depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires methods for perturbing gene expression with similar precision. Emerging chemical technologies can enable such perturbations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to photo-inactivate genes in zebrafish embryos with spatiotemporal control. This chapter describes general principles for cMO design and methods for cMO assembly in three steps from commercially available reagents. Experimental techniques for the microinjection and photoactivation of these reagents are described in detail, as well as the preparation and application of caged fluorescein dextran (cFD) for labeling irradiated cells. Using these protocols, cMOs can be effective tools for functional genomic studies in zebrafish and other model organisms.
View details for DOI 10.1016/B978-0-12-374814-0.00009-4
View details for Web of Science ID 000295551800009
View details for PubMedID 21924162
View details for PubMedCentralID PMC4408312
Lineage labeling of zebrafish cells with laser uncagable fluorescein dextran.
Journal of visualized experiments : JoVE
A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling and pressure injection of traceable enzymes to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularity attractive tools for lineage labeling and to observe the migration of cells in vivo. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequently, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.
View details for DOI 10.3791/2672
View details for PubMedID 21559005
Synthetic Strategies for Studying Embryonic Development
CHEMISTRY & BIOLOGY
2010; 17 (6): 590-606
Developmental biology has evolved from a descriptive science to one based on genetic principles and molecular mechanisms. Although molecular biology and genetic technologies have been the primary drivers of this transformation, synthetic strategies have been increasingly utilized to interrogate the mechanisms of embryonic patterning with spatial and temporal precision. In this review, we survey how chemical tools and engineered proteins have been used to perturb developmental processes at the DNA, RNA, protein, and cellular levels. We discuss the design principles, experimental capabilities, and limitations of each method, as well as future challenges for the chemical and developmental biology communities.
View details for DOI 10.1016/j.chembiol.2010.04.013
View details for Web of Science ID 000279616700007
View details for PubMedID 20609409
View details for PubMedCentralID PMC2902240
Oligonucleotide-Based Tools for Studying Zebrafish Development
2010; 7 (1): 31-40
Synthetic and nonnatural oligonucleotides have been used extensively to interrogate gene function in zebrafish. In this review, we survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression. We also examine recent strategies for achieving spatiotemporal control of oligonucleotide function, particularly light-gated technologies that exploit the optical transparency of zebrafish embryos.
View details for DOI 10.1089/zeb.2010.0650
View details for Web of Science ID 000277029700005
View details for PubMedID 20392138
View details for PubMedCentralID PMC2860060
Controlling Destiny through Chemistry: Small-Molecule Regulators of Cell Fate
ACS CHEMICAL BIOLOGY
2010; 5 (1): 15-34
Controlling cell fate is essential for embryonic development, tissue regeneration, and the prevention of human disease. With each cell in the human body sharing a common genome, achieving the appropriate spectrum of stem cells and their differentiated lineages requires the selective activation of developmental signaling pathways, the expression of specific target genes, and the maintenance of these cellular states through epigenetic mechanisms. Small molecules that target these regulatory processes are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming. Pharmacological modulators of cell fate could also help remediate human diseases caused by dysregulated cell proliferation or differentiation, heralding a new era in molecular therapeutics.
View details for DOI 10.1021/cb900249y
View details for Web of Science ID 000273653800002
View details for PubMedID 20000447
View details for PubMedCentralID PMC2807212
Versatile Synthesis and Rational Design of Caged Morpholinos
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2009; 131 (37): 13255-13269
Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.
View details for DOI 10.1021/ja809933h
View details for Web of Science ID 000270186500036
View details for PubMedID 19708646
View details for PubMedCentralID PMC2745229
Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (33): 14132-14137
Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.
View details for DOI 10.1073/pnas.0907134106
View details for Web of Science ID 000269078700091
View details for PubMedID 19666565
View details for PubMedCentralID PMC2721821
Converse Conformational Control of Smoothened Activity by Structurally Related Small Molecules
JOURNAL OF BIOLOGICAL CHEMISTRY
2009; 284 (31): 20876-20884
The seven-pass transmembrane protein Smoothened (Smo) is an essential component of the Hedgehog (Hh) signaling pathway that is critically involved in normal animal development as well as pathological malignancies. In studying Hh-related biological processes, it would be highly desirable if Smo activity could be instantly switched between activation and inhibition. Using Gli1-dependent GFP transgenic zebrafish and in vitro biochemical assays, we identified and characterized two potent Smo inhibitors, SANT74 and 75 (Smoothened antagonist 74 and 75), by screening a small molecule library designed based on the scaffold of Smo agonist SAG. These compounds are structural analogs of SAG with the methyl group substituted by a propyl or allyl group in SANTs. We show that SANTs and SAG exert opposite effects on Smo activity by regulating protein conformation. Our study represents the first demonstration of conformational regulation of Smo by small molecule analogs, and the combinational use of these Smo modulators in a temporal controlled fashion should be useful for studying Hh biology.
View details for DOI 10.1074/jbc.M807648200
View details for Web of Science ID 000268316100051
View details for PubMedID 19366682
View details for PubMedCentralID PMC2742853
Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent
2009; 328 (2): 342-354
Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.
View details for DOI 10.1016/j.ydbio.2009.01.036
View details for Web of Science ID 000265239100015
View details for PubMedID 19389352
View details for PubMedCentralID PMC2674121
A small molecule that binds Hedgehog and blocks its signaling in human cells
NATURE CHEMICAL BIOLOGY
2009; 5 (3): 154-156
Small-molecule inhibition of extracellular proteins that activate membrane receptors has proven to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic hedgehog (Shh) protein and blocks Shh signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.
View details for DOI 10.1038/nchembio.142
View details for Web of Science ID 000263556100012
View details for PubMedID 19151731
View details for PubMedCentralID PMC2770933
The Imidazopyridine Derivative JK184 Reveals Dual Roles for Microtubules in Hedgehog Signaling
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2009; 48 (13): 2321-2324
Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).
View details for DOI 10.1002/anie.200805666
View details for Web of Science ID 000264784400011
View details for PubMedID 19222062
- Targeted and Conditional Gene Expression Workshop, 8th International Conference on Zebrafish Development and Genetics. Zebrafish 2008; 5 (3): 193-195
The decoupling of Smoothened from G alpha(i) proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning
2008; 321 (1): 188-196
The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Galpha(i) proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Galpha(i) protein activity appears to significantly reduce Hh pathway activity in Ptc(-/-) mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Galpha(i) protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Galpha(i) protein, Galpha(i2)QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Galpha(i) activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.
View details for DOI 10.1016/j.ydbio.2008.06.014
View details for Web of Science ID 000258710800016
View details for PubMedID 18590719
View details for PubMedCentralID PMC2597282
- Fish 'n clicks NATURE CHEMICAL BIOLOGY 2008; 4 (7): 391-392
Gene regulation technologies in zebrafish
2008; 4 (4): 300-308
Achieving the potential of zebrafish models in biomedical research is contingent on the development of reverse-genetic resources. This review describes current technologies for genetic perturbations in zebrafish, including an ecdysone receptor-based system that permits conditional transgene expression. Such methodologies promise to enable new zebrafish models for interrogating human physiology and disease.
View details for DOI 10.1039/b718447f
View details for Web of Science ID 000254162200004
View details for PubMedID 18354783
Chemical technologies for probing embryonic development
CHEMICAL SOCIETY REVIEWS
2008; 37 (7): 1294-1307
Embryogenesis is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. Understanding this process at the molecular and systems levels will require an interdisciplinary approach, including the concepts and technologies of chemical biology. This tutorial review provides an overview of chemical tools that have been used in developmental biology research, focusing on methods that enable spatiotemporal control of gene function and the visualization of embryonic patterning. Limitations of current approaches and future challenges are also discussed.
View details for DOI 10.1039/b703023c
View details for Web of Science ID 000257018800002
View details for PubMedID 18568156
View details for PubMedCentralID PMC2531163
Light-controlled gene silencing in zebrafish embryos
NATURE CHEMICAL BIOLOGY
2007; 3 (10): 650-651
Functional genomic studies in zebrafish frequently use synthetic oligonucleotides called morpholinos that block RNA splicing or translation. However, the constitutive activity of these reagents limits their experimental utility. We report here the synthesis of a photoactivatable morpholino targeting the no tail (ntl) gene. This caged reagent permits spatiotemporal gene regulation in vivo and the photochemical generation of functionally mosaic organisms.
View details for DOI 10.1038/nchembio.2007.30
View details for Web of Science ID 000249642700013
View details for PubMedID 17717538
Small-molecule regulation of zebrafish gene expression
NATURE CHEMICAL BIOLOGY
2007; 3 (3): 154-155
The zebrafish has emerged as a versatile model organism for biomedical research, yet its potential has been limited by a lack of conditional reverse-genetic tools. Here we report a chemically inducible gene expression technology that has orthogonality to vertebrate signaling processes, high induction levels, and rapid kinetics. Coupled with tissue-specific promoters, this system provides multidimensional control of gene expression and will enable new models of human disorders and diseases.
View details for DOI 10.1038/nchembio858
View details for Web of Science ID 000244336600010
View details for PubMedID 17237798
Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (20): 7550-7560
Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.
View details for DOI 10.1128/MCB.00546-06
View details for Web of Science ID 000241252300018
View details for PubMedID 16908539
View details for PubMedCentralID PMC1636865
Purmorphamine activates the Hedgehog pathway by targeting Smoothened
NATURE CHEMICAL BIOLOGY
2006; 2 (1): 29-30
Hedgehog (Hh) signaling is an important regulator of embryonic patterning, tissue regeneration, stem cell renewal and cancer growth. A purine derivative named purmorphamine was previously found to activate the Hh pathway and affect osteoblast differentiation through an unknown mechanism. We demonstrate here that purmorphamine directly targets Smoothened, a critical component of the Hh signaling pathway.
View details for DOI 10.1038/nchembio.753
View details for Web of Science ID 000233971600010
View details for PubMedID 16408088
Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2
2004; 306 (5705): 2257-2260
Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.
View details for DOI 10.1126/science.1104135
View details for Web of Science ID 000225950000047
View details for PubMedID 15618519
Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened
GENES & DEVELOPMENT
2002; 16 (21): 2743-2748
The steroidal alkaloid cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to vertebrate Hedgehog signaling. We show here, using photoaffinity and fluorescent derivatives, that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo.
View details for DOI 10.1101/gad.1025302
View details for Web of Science ID 000179027900002
View details for PubMedID 12414725
Small molecule modulation of Smoothened activity
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (22): 14071-14076
Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling.
View details for DOI 10.1073/pnas.182542899
View details for Web of Science ID 000178967400021
View details for PubMedID 12391318
Medulloblastoma growth inhibition by Hedgehog pathway blockade
2002; 297 (5586): 1559-1561
Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.
View details for Web of Science ID 000177697300059
View details for PubMedID 12202832
Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine
2000; 406 (6799): 1005-1009
Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.
View details for Web of Science ID 000089020200048
View details for PubMedID 10984056
The identification of myriocin-binding proteins (vol 6, pg 221, 1999)
CHEMISTRY & BIOLOGY
1999; 6 (6): R186-R186
View details for Web of Science ID 000084001000010
Crystal structure of PI3K SH3 domain at 2.0 angstrom resolution
JOURNAL OF MOLECULAR BIOLOGY
1996; 257 (3): 632-643
The P13K SH3 domain, residues 1 to 85 of the P1-3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space group P4(3)2(1)2 diffract to 2.0 angstroms resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact beta barrel with an over-all confirmation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein-protein aggregation that could be mediated by SH3 domains.
View details for Web of Science ID A1996UC77300015
View details for PubMedID 8648629
Protein Structure-Based Combinatorial Chemistry: Discovery of Non-Peptide Binding Elements to Src SH3 Domain
J. Am. Chem. Soc.
1996; 118 (1): 287-288
View details for DOI 10.1021/ja953750v
COMBINATORIAL SYNTHESIS AND MULTIDIMENSIONAL NMR-SPECTROSCOPY - AN APPROACH TO UNDERSTANDING PROTEIN-LIGAND INTERACTIONS
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION IN ENGLISH
1995; 34 (9): 953-969
View details for Web of Science ID A1995RB00200001
2 BINDING ORIENTATIONS FOR PEPTIDES TO THE SRC SH3 DOMAIN - DEVELOPMENT OF A GENERAL-MODEL FOR SH3-LIGAND INTERACTIONS
1994; 266 (5188): 1241-1247
Solution structures of two Src homology 3 (SH3) domain-ligand complexes have been determined by nuclear magnetic resonance. Each complex consists of the SH3 domain and a nine-residue proline-rich peptide selected from a large library of ligands prepared by combinatorial synthesis. The bound ligands adopt a left-handed polyproline type II (PPII) helix, although the amino to carboxyl directionalities of their helices are opposite. The peptide orientation is determined by a salt bridge formed by the terminal arginine residues of the ligands and the conserved aspartate-99 of the SH3 domain. Residues at positions 3, 4, 6, and 7 of both peptides also intercalate into the ligand-binding site; however, the respective proline and nonproline residues show exchanged binding positions in the two complexes. These structural results led to a model for the interactions of SH3 domains with proline-rich peptides that can be used to predict critical residues in complexes of unknown structure. The model was used to identify correctly both the binding orientation and the contact and noncontact residues of a peptide derived from the nucleotide exchange factor Sos in association with the amino-terminal SH3 domain of the adaptor protein Grb2.
View details for Web of Science ID A1994PT13200054
View details for PubMedID 7526465
SH3 DOMAIN-MEDIATED DIMERIZATION OF AN N-TERMINAL FRAGMENT OF THE PHOSPHATIDYLINOSITOL 3-KINASE P85 SUBUNIT
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
1994; 4 (14): 1755-1760
View details for Web of Science ID A1994NY39500019
STRUCTURAL BASIS FOR THE BINDING OF PROLINE-RICH PEPTIDES TO SH3 DOMAINS
1994; 76 (5): 933-945
A common RXL motif was found in proline-rich ligands that were selected from a biased combinatorial peptide library on the basis of their ability to bind specifically to the SH3 domains from phosphatidylinositol 3-kinase (PI3K) or c-Src. The solution structure of the PI3K SH3 domain complexed to one of these ligands, RKLPPRPSK (RLP1), was determined. Structure-based mutations were introduced into the PI3K SH3 domain and the RLP1 ligand, and the influence of these mutations on binding was evaluated. We conclude that SH3 domains recognize proline-rich motifs possessing the left-handed type II polyproline (PPII) helix conformation. Two proline residues directly contact the receptor. Other prolines in the ligands appear to function as a molecular scaffold, promoting the formation of the PPII helix. Three nonproline residues consisting of combinations of arginine and leucine interact extensively with the SH3 domain and appear to confer ligand specificity.
View details for Web of Science ID A1994NA89000018
View details for PubMedID 7510218
Affinity Capillary Electrophoresis: Insights into the Binding of SH3 Domains by Peptides Derived from an SH3-Binding Protein
J. Org. Chem.
1994; 59 (10): 2885-2886
View details for DOI 10.1021/jo00089a038
BIASED COMBINATORIAL LIBRARIES - NOVEL LIGANDS FOR THE SH3 DOMAIN OF PHOSPHATIDYLINOSITOL 3-KINASE
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1993; 115 (26): 12591-12592
View details for Web of Science ID A1993MQ10000051
AFFINITY ELECTROPHORESIS IN MULTISECTIONAL POLYACRYLAMIDE SLAB GELS IS A USEFUL AND CONVENIENT TECHNIQUE FOR MEASURING BINDING CONSTANTS OF ARYL SULFONAMIDES TO BOVINE CARBONIC ANHYDRASE-B
1993; 65 (10): 1314-1322
This paper describes convenient preparations of heterogeneous multisectional polyacrylamide slab gels and the protocols that use these gels to measure protein-ligand binding constants [using bovine carbonic anhydrase B (CAB) as a model system]. Unlike procedures for affinity electrophoresis using tube gels, all binding information concerning protein-ligand interactions was encoded in a single multisectional gel: the procedure involving for measuring binding constants required no postelectrophoresis manipulation of gels. Use of these types of gels improves the accuracy of affinity gel electrophoresis (AGE) by providing reliable internal protein standards. Binding constants measured by AGE agree with those determined in homogeneous solution by spectrophotometric measurements. This technique has been used to investigate the influence of the length of the spacer separating the ligand and the polyacrylamide backbone on the binding constants. Dissociation constants obtained using the affinity gels approach the values measured in free solution, when the spacer is sufficiently long (> or = 18 A); affinity ligands having short spacers give high apparent dissociation constants.
View details for Web of Science ID A1993LC09800005
View details for PubMedID 8517543
Molecular recognition in gels, monolayers, and solids
Macromolecular Assemblies in Polymeric Systems
American Chemical Society. 1992: 228–239
View details for DOI 10.1021/bk-1992-0493.ch019